Allogeneic hematopoietic cell transplant (AlloHCT) is used to treat cancer and is proposed as a method to achieve donor-specific tolerance after organ transplantation. Inducing tolerance to an allogeneic transplant (Tx) would alleviate the need for immunosuppressants, which prevent Tx rapid rejection by globally blunting host immune responses. Unfortunately, these drugs promote infection, cancer, and kidney damage, and fail to prevent chronic rejection, a progressive alloimmune-driven fibrotic process that culminates in Tx dysfunction and failure. The use of AlloHCT for Tx tolerance is limited by the potential for graft-versus-host disease (GVHD), an often fatal condition where donor immune cells attack the host tissues. Thus far, AlloHCT protocols have failed to induce durable tolerance in patients. To address these CLINICAL PROBLEMS, innovation in recipient treatment is needed, and our OBJECTIVE is to use cutting-edge transgenic mouse models to establish the means to eliminate GVHD risks and provide an immunological environment supporting robust Tx-specific tolerance induction. To create space for donor stem cells and prevent their rapid rejection by host immune cells, AlloHCT protocols remove recipient immune and bone marrow cells with harsh conditioning regimens involving total body irradiation (TBI) or chemotherapy. These treatments unavoidably cause tissue damage releasing alarmins, or self-derived immunomodulatory molecules that activate the cells of the immune system. Interleukin (IL)-33 is a proposed alarmin and epithelial cell-derived cytokine, which may drive GVHD and modulate tolerance after AlloHCT. Our preliminary data reveal that conditioning regimens augment IL-33 in GVHD-target tissue and IL-33-stimulated alloreactive donor T cells mediate GVHD. We also demonstrate that preconditioning of AlloHCT recipients with IL-33 before TBI paradoxically protects against GVHD development. These data are consistent with pleiotropic functions of IL-33, which can promote both Type 1 and Type 2 pro-inflammatory responses, but also expand suppressive regulatory T cells. We HYPOTHESIZE that the pleiotropic functions of molecules like IL-33 can be precisely controlled to shape alloimmunity. To precisely control IL-33, we must identify the mechanisms modulating IL-33 expression, release, and promotion of alloimmunity after AlloHCT (AIM 1). We will also assess if countering proposed drivers of IL-33 pro-inflammatory properties after AlloHCT will prevent GVHD and promote Tx tolerance (AIM 2). The proposed studies are INNOVATIVE because they could establish a new paradigm in transplantation where pleiotropic immune stimuli are freed to support tolerance by countering their pro-inflammatory triggers or augmenting their regulatory properties. These studies are SIGNIFICANT, as they will increase our understanding of IL-33 functions in GVHD and may eliminate it as a potential risk factor following AlloHCT. AlloHCT-induced tolerance would eliminate chronic rejection and immunosuppressant-related side effects, thus turning solid organ Tx into a life saving, not just a life extending procedure for end-stage organ failure patients.